Brucella abortus antigen omp25 vaccines: Development and targeting based on Lactococcus lactis

Abstract Background Most Brucella infections take place on mucosal membranes. Therefore, creating vaccinations delivered through the mucosa may be crucial for managing brucellosis. Consequently, we assessed the efficacy of a recombinant oral antigen delivery system based on Lactococcus lactis for Brucella abortus omp25 antigen. Method Oral vaccinations with L. lactis transformed with pNZ8148 variants encoding for omp25 (pNZ8148:omp25) and free‐pNZ8148 were administered to mice. On day 30, following immunization in animal groups, anti‐omp25‐specific IgG1 antibodies were assessed by the ELISA test. Additionally, nasal and bronchoalveolar lavages containing omp25‐specific secretory IgA (sIgA) were analysed by ELISA. ELISA test and real‐time PCR were also used to analyse cytokine responses up to 28 days following the last boost. In addition, the protective potential of L. lactis pNZ8148:omp25 vaccines was assessed in BALB/c mice by exposing them to the B. abortus strain. Results Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. The produced plasmid was digested using KpnI and XbaI. By electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombinant plasmid, was confirmed. Antigen expression at the protein level revealed that the target group generated the 25 kDa‐sized omp25 protein, but there was no protein expression in the control group. Fourteen days after priming, there was a considerable amount of omp25‐specific IgG1 in the sera of mice vaccinated with pNZ8148–Usp45–omp25–L. lactis (p < 0.001 in target groups compared to the phosphate‐buffered saline control group). IFN‐γ and TNF‐α levels were more significant in samples from mice that had been given the pNZ8148–Usp45–omp25–L. lactis and IRBA vaccinations, in samples taken on days 14 and 28, respectively (p < 0.001). The pNZ8148–Usp45–omp25–L. lactis and IRBA immunization groups had significantly greater IL‐4 and IL‐10 transcription levels than the other groups. The spleen portions from the pNZ8148–Usp45–omp25–L. lactis and IRIBA vac group had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process. Conclusion Our study offers a novel method for using the food‐grade, non‐pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25. This method offers an appealing new approach to assessing the cost‐effective, safe, sustainable, simple pilot development of pharmaceutical products.


INTRODUCTION
Brucellosis is an endemic illness spread widely worldwide by domesticated animals infected with Brucella spp. (Franc et al., 2018). Specifically, Brucella melitensis and Brucella abortus are the major causes. The most common ways to get brucellosis are eating, breathing or being close to animal bites or sores (Franc et al., 2018;Ghajari et al., 2021).
Like other naturally occurring intracellular infections, Brucella can exist outside of eukaryotic cells but needs to infect and proliferate inside living things to thrive (Hosseini et al., 2022). Because Brucella is so effectively suited to the intracellular environment, it should be referred to as a facultative extracellular intracellular parasite (Pizarro-Cerdá et al., 2000). This bacterium causes feverish illnesses in people called 'undulant fever' and abortions in cattle, goats and sheep, creating a burden for both the economy and the general health problem (Jamil et al., 2021). Tolerance to Brucella relies on accumulated cell-mediated immunity (CMI), just as tolerance to other facultative intracellular bacterial infections (Saxena et al., 2018). Most research on the immune reaction to Brucella spp. has been done on mice. The protective immunity in this mouse model appears to be controlled by both cellular and humoral effector pathways Dorneles, Teixeira-Carvalho, et al., 2015).
An immunological reaction mediated by CD4+ and CD8+ T lymphocytes is particularly critical to managing the pathogen, according to in vivo evaluation. Gamma interferon-producing CD4+ T cells within these lymphocyte subtypes are crucial for the immune system's ability to recover from infection (Reschner et al., 2008). Live attenuated variants of B. abortus, such as variant RB51 or variant 19, are used in the vaccination vs. brucellosis in livestock and wild ruminants. Although variant 19 effectively triggers a protective immune system response, it harms humans and causes abortion when given to pregnant cattle (Ribeiro et al., 2002). Even though variant RB51 is the preferred brucellosis vaccination for cattle is produced from the virulent B. abortus 2308 variant and is not, like strain 19, advised for pregnant animals Dorneles, Teixeira-Carvalho, et al., 2015;Kargar et al., 2014). Additionally, as there are presently no human vaccines on the market, a different strategy to immunize humans and animals against brucellosis would be to utilize a foodgrade oral vaccine expressing a shared protein from B. melitensis and B. abortus (Sáez et al., 2012). melitensis Rev1 and B. abortus S19 and RB51. These vaccine formulations are far from ideal because they have various drawbacks, such as the fact that they are harmful for human use and might cause miscarriage in pregnant calves. These vaccines are also regarded as too virulent or dangerous for animal use (Rezaei et al., 2020). Antigen injection by live lactic acid bacteria (LAB) as food-grade, non-invasive and non-pathogenic vaccines is a promising immunization approach . This strategy might eliminate possible drawbacks of using live B. abortus isolates as protein delivery vehicles, such as interference with diagnostic testing, residual pathogenicity and reversion hazard (Heidary et al., 2022). It offers a widespread and affordable vaccine administration method. An effective immune system response has been demonstrated following mucosal vaccination using LAB, which has been genetically engineered to generate bacterial and viral proteins (Levit et al., 2022).
These positive outcomes suggest the viability of the LAB-based vaccination strategy. The potential to concurrently produce humoral and cell-mediated immune function at mucosal locations and throughout the body is another benefit of mucosal-delivered vaccinations (Taghinezhad et al., 2021). Non-invasive, non-pathogenic Lactococcus lactis is a potential antigen delivery device for mucosal vaccination inside LAB (Bermúdez-Humarán et al., 2011;Piri-Gharaghie, Beiranvand, et al., 2022;Piri-Gharaghie, Doosti, et al., 2022). The digestive tract will not get colonized by L. lactis, even though it may survive transit through the gastrointestinal system (Daniel et al., 2013). L. lactis expression systems have been developed during the past 10 years due to gene engineering.
Recombinant isolates of L. lactis have been demonstrated to generate specific immunological reactions in mice (Plavec et al., 2021). Viral, bacterial and parasite antigens have been produced in L. lactis. Additionally, IL-12 (IL-12, a cytokine aid in fighting against several bacterial, viral and parasite infections) has been made using L. lactis (Zeinali et al., 2022).
The discovery of immunogenic proteins that may elicit a cellmediated immune response, which is necessary to fight the pathogen's intracellular location, has been the foundation of current techniques for developing novel vaccines vs. B. abortus Piri-Gharaghie, Doosti, et al., 2022). The omp25, the antigen of B. abortus, can boost the immune defence .
Mice that received immunization with pure omp25 showed exceptional protection against B. abortus infection . This study examines the possibility of using L. lactis to treat mice with B. abortus omp25 as a vaccine.

Bioinformatics analyses: screening of vaccine candidate proteins
In the identification of a possible vaccination candidate for this investigation, 35 genome sequences of B. abortus strains were found using the Vaxign website (http://www.violinet.org/vaxign/). Candidates for vaccines were selected from the 10 gene-coding proteins research participants. The requirements were: • A threshold of one transmembrane helix (≤1).
• A possibility of adhesion higher than 0.51.
• A lack of similarity to human or mouse proteins.
The vaccine candidate components must not interact with human or mouse biomolecules to reduce the likelihood of a host cell interacting with the vaccination. The proteins were examined for this purpose using the host protein Homo sapiens/mice and the BLASTp web service on the NCBI website. The proteins from the B. abortus genus will be utilized for further investigation if the BLASTp web service confirms the protein specificity. The existence of various proteins in the B. abortus proteome was examined using the UniProt database (http:// www.ncbi.nlm.nih.gov/protein), and the NCBI saved the sequence of amino acids in FASTA format for subsequent research. To establish the exact location of proteins in the B. abortus bacteria, the CELLO program (http://cello.life.nctu.edu.tw/) has been used. It can identify the location of molecules outside the cell with a Localization Reliability ≥1.5 on the outer or inner membrane and determine if the protein is cytoplasmic or periplasmic. The VaxiJen software (http://www.ddg-pharmfac. net/vaxijen/VaxiJen/VaxiJen.html) was used to calculate the antigenicity of the selected proteins, using a cut-off of 0.6. The physicochemical characteristics of the B. abortus outer membrane proteins (OMPs) were examined using a bioinformatics method. All potential dominant Bcell and T-cell epitopes were then predicted. Optimum Antigen Design Tool, a piece of bioinformatics software, was used to anticipate the B-cell epitopes (GenScript, China). The secondary structure, surface availability, solubility in water, flexibility and antigenic index of Bcell epitopes were anticipated. The A and E subregions of the mouse MHC-II genes were analysed and predicted using the Immune Epitope Database Analysis Resource (IEDB) (https://tools.iedb.org/mhci/) when determining T-cell epitopes. As a potential vaccination candidate, the proteins with more than five B-cell and five T-cell epitopes were screened (Piri-Gharaghie, Doosti, et al., 2023).

Animals
Female BALB/c mice (6-8-week old) were purchased from the Biotechnology Research Animal Laboratory Center. Following the guidelines of the Ethics Committee, mice were maintained in specified pathogenfree environments. All animal protocols were carried out following the regulations established by the Institutional Animal Care and Use Committee.

Positive control, bacterial strains and growth conditions
The E. coli TOP10F and L. lactis IBRC-M 11051 isolates were purchased. L. lactis isolates were cultivated in M17 medium (Ibrsco, Iran) at 30 • C and in anaerobic condition with a 1% glucose supplement. E. coli TOP10F isolates were cultivated at 37 • C in Luria-Bertani media.

Recombinant plasmid construction
60 183 The following antibiotic concentrations were used to select plasmids: 100 μg/mL of ampicillin for E. coli and 5 μg/mL of chloramphenicol for L. lactis.

Confirmation of the cloning of omp25 into the pNZ8148 vector
The PCR reaction was conducted to monitor omp25 using specific primers (Table 1). A 20-mL PCR reaction contains 2 mL of ×10 PCR buffer (Yekta Tajhiz Azma, Iran), 2 mM MgCl 2 , 200 μM dNTPs (Yekta Tajhiz Azma, Iran), 10 pmol of each primer (CinnaGen, Iran), 100 ng of plasmid DNA and 1 unit of Taq DNA polymerase enzyme (Yekta Tajhiz Azma, Iran). The PCR temperature protocol consists of an initial annealing step at 95 • C for 5 min, followed by 30 repeated stages at 94 • C for 1 min, 52 • C for 1 min and 72 • C for 1 min. Finally, the final elongation was performed at 72 • C for 5 min. The PCR product was electrophoresed on a 1% agarose gel containing ethidium bromide, and the bands were observed and recorded with a UVITech (England) gel imaging device. The recombinant vector was subjected to enzyme digestion and sequencing by GENEray company, employing the restriction enzymes KpnI and XbaI.

Expression and immunodetection of omp25
The recombinant L. lactis organism carrying pNZ8148:omp25 was used to culture fresh medium overnight at a dilution of 1/100 to induce the nisin promoter. The colonies were cultured for 1 h at an optical density of 600 nm (OD600) of 0.6 (∼0.6) before cell fractionation and protein identification. Nisin-induced L. lactis pNZ8148:omp25 was obtained by centrifuging at 12,000 × g for 10 min at 4 • C, concentrating the bacterial protein supernatants by 50 times relative to their original volume, and then analysing it using 12% sodium dodecyl sulphatepolyacrylamide gel electrophoresis before electrotransferred it onto the nitrocellulose membrane. The membranes were treated with a mouse IgG monoclonal antibody anti-omp25 (Rozhan AZMA, Iran)

Grouping of animals and schedule for oral immunization
In this investigation, 120 female BALB/c mice aged 6-8 weeks and weighing 15-20 g were employed. Animals have injected pNZ8148-omp25 through oral immunization (100 μg of pDNA). A total of 80 mice were split into 5 groups of 20, whereas an additional 20 mice served as negative controls (n = 20) and received PBS. The quantity of mice used in each group is shown in Table 2. The control and recombinant strains of L. lactis pNZ8148:omp25 were cultivated as described earlier and stimulated for 1 h with nisin. An oral pipette was used to administer 10 8 L. lactis pNZ8148:omp25 colony forming unit (CFU) three times to a group of 20 mice. The treatment was given over 3 days (days 0-2,

ELISA analysis
Two days before each inoculation and 15 days after the final vaccination, mice were bled, and serum samples were collected (five mice per group). The substance was gathered and kept at −70 • C. By using an indirect ELISA following a predetermined procedure, the presence of sera G1 (IgG1) and secretory IgA (sIgA) from nasal lavages with specificity to omp25 was identified. Purified romp25 was collected, diluted to 2 μg/mL in carbonate-bicarbonate buffer (pH 9.6) and utilized to coat the wells of a polystyrene plate with crude Brucella proteins, an extract acquired from organisms treated with a hyperosmotic salt solution and sonication, at a concentration of 10 μg/mL within the same buffer (Nunc-Immuno plate with MaxiSorp surface). Isotype-specific goat anti-mouse horseradish omp25 conjugates were administered at a dilution of 1:1000. The average specific OD450 for 10 sera from nonimmunized animals evaluated at a concentration of 1:50 was multiplied by the standard deviation to get the assay's cut-off ratio. The data were normalized and represented as the endpoint titre OD of antigenspecific sIgA concerning 1 μg of total IgA in the sample to account for differences in the effectiveness of recovering secretory antibodies among individuals. Total IgG1 values are shown as the mean OD450 at a plasma dilution of 1:100.

2.9
Detection of cellular immune response

Challenge experiments
The protection experiments were carried out following Piri-Gharaghie et al. A total of 10 mice from each group were exposed to 10 4 CFU of B.
abortus strain 2308 intraperitoneally 2 weeks after the previous dose.
The mice were tracked for 7 days, and each group's body weight, clinical score and survival rate were recorded every day. Each mouse's overall clinical sign was rated on a range of 0 to −5 on a sliding scale. Individual clinical ratings ranged from 0 (normal, active, healthy), −1 (slightly sick, slightly ruffled fur, otherwise normal), −2 (ill, ruffled fur, sluggish movement, hunching), −3 (extremely sick, ruffled hair, prolonged movement, stooped, eyes shut), −4 (moribund) and −5 (dead

Histopathological examination
Livers were extracted aseptically and fixed in 10% formalin. After embedding in paraffin, slices were examined histopathologically under a microscope after staining with haematoxylin and eosin. The proportion of the lesion area among the whole liver area was used to assess liver damage using an ImagePro macro.

Statistical analyses
GraphPad Prism 5.0 was used to examine the data and perform statistical tests. A one-way analysis of variance was used to compare means, followed by a Tukey-Kramer post hoc test with a 95% confidence interval. A Chi-square test with Yates' correction was used to compare the survival rates between immunized mice and the control group. Differences were considered significant at p < 0.05 and p < 0.01.

A potential vaccine candidate has been discovered
The Vaxign database identified 44 proteins with top qualities. Then, 18 hypothetically pathogenic proteins were selected. The potential vaccine candidates were reduced to 6 after protein antigenicity was evaluated. After the UniProt dataset confirmed the existence of six proteins in B. abortus, the access number of these proteins was chosen on the NCBI website. They were categorized as extracellular, outer membrane, periplasmic, inner membrane and cytoplasmic pro-  Table 3. Based on the initial screening results, the omp25 protein was identified for immunogenicity because it had the maximum solubility and flexibility and antigenic values of 0.75. Based on the screening results, the omp25 protein was identified as immunogenic because it has maximum solubility, which helps the protein reactivity with immune cells. Moreover, omp25 is more flexible than other proteins. This leads to immune cells recognizing different epitopes of this protein in different spatial structures. On the other hand, omp25 had higher antigen values (0.75) than other proteins, which indicates that it has a high potential to activate immune cells. The prospective vaccine candidate omp25 showed B-cell linear epitopes with a value of 0.5 that are disseminated with the proteins, according to IEDB research. Omp25 had five B-cell epitopes and five T-cell epitopes. The quantity of surface-exposed conformational B-cell epitopes is shown in Figure 1.

Generating and identifying recombinant pNZ8148-Usp45-omp25-L. lactis
The DNA vaccine construct was constructed by inserting the omp25 gene into the expression vector pNZ8148. The Genetic code of the recombinant plasmid served as a measure of the cloning effectiveness.
Additionally, DNA analysis showed that the recombinant plasmid's virulence gene sequence was 100% identical to that of the B. abortus bacterium. The produced plasmid was digested using KpnI and XbaI. By the electrophoretic isolation of the digestion fragments at 786 bp, the omp25 gene, the successful production of the recombi-

3.3
Expression of recombinant pNZ8148-Usp45-omp25-L. lactis in mRNA and secretion of omp25 in L. lactis The relative amounts of mRNA expression of the omp25 Gene delivery were determined using reverse transcriptase-PCR. The 786 bp band on the agarose gel shows that the transcription of the gene has taken place ( Figure 2B). The Western blot analysis by Rojan Azma also revealed that the specific proteins were generated at the protein level. Antigen expression at the protein level revealed that the target group generated the 25 kDa-sized omp25 protein, but there was no protein expression in the control group ( Figure 2C)

Immune responses induced by oral immunization
A prospective vaccination method involved employing non-pathogenic LAB (L. lactis) as target delivery vehicles to induce humoral and cellmediated immune responses throughout the body, including mucosal sites. Thus, we chose to evaluate whether vaccination with L. lactis, which has been genetically engineered to release omp25, can induce a particular immune response in various groups, as shown in Table 2.
After oral immunization, BALB/c mice were tested for omp25-specific IgG1 or sIgA using an indirect ELISA. As seen in Figure 3A, 14 days after priming, there was a considerable amount of omp25-specific IgG1 in the sera of mice vaccinated with pNZ8148-Usp45-omp25-L. lactis (p < 0.001 in target groups compared to the PBS control group). Similar substantial values were seen at day 28, although the levels returned to baseline compared to the PBS negative control groups. Compared to the values from mice in the PBS control group, the findings revealed 14 days after vaccination with pNZ8148-Usp45-omp25-L. lactis, levels of omp25-specific IgG1 were significantly ( Figure 3A; p < 0.0001) higher in both pNZ8148-Usp45-omp25-L. lactis and IRBA vaccine (positive control) groups. Even while the levels of IgG1 decreased on day 28 compared to controls, they were still considerably greater. The levels of total IgG in sera from animals that had received the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccines were also higher than those from the negative control groups in samples obtained on days 14 and 28, respectively (p < 0.001) ( Figure 3C). At day 28, the levels of total IgG in the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccination groups were remarkably comparable, although they were still considerably more significant when compared to the control groups (p < 0.001).
We also assessed the induction of mucosal reactions in animals that had received vaccinations. Omp25-specific sIgA titres were measured for this purpose using bronchoalveolar lavages and nasal swabs ( Figure 3B). According to the findings, vaccinated mice produced more antigen-specific mucosal IgA than PBS-treated control mice or other groups (p < 0.05). Interestingly, compared to animals inoculated with control groups, mice were vaccinated with pNZ8148-Usp45-omp25-L. lactis displayed substantially higher sIgA titres in nasal lavages (p < 0.05). In contrast, IgA titres in the pNZ8148-Usp45-omp25-L.
lactis and IRBA vaccination groups were comparable and considerably higher than those in the PBS control group (p < 0.05).

Cytokine secretion
After re-stimulation, cytokine production from spleen cells was examined in vitro. When challenged, splenocytes from immunized and non-immunized mice were produced and grown with various antigens (Table 2). Unstimulated cells from animals administered PBS, pNZ8148, L. lactis and pNZ8148-L. lactis showed no significant changes (p > 0.05).
On the other hand, unimmunized (saline inoculation) mouse cells failed to substantially boost their IFN-γ and TNF-α production with restimulation (p > 0.05). After 14 days, cells from mice were vaccinated with pNZ8148-Usp45-omp25-L. lactis, and IRBA secreted more IFNγ and TNF-α (Figure 4). IFN-γ and TNF-α levels were more significant in samples from mice that had been given the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccinations, as well as the negative control groups in samples taken on days 14 and 28, respectively (p < 0.001) (Figure 4).
Compared to the control groups, the concentrations of IFN-γ and TNF-α in the pNZ8148-Usp45-omp25-L. lactis and IRBA vaccination groups at day 28 were impressively identical (p < 0.001). IFN-γ and TNF-α concentrations were still significantly higher on day 28 despite a decline compared to controls. IL-4 and IL-10 inductions in mice that had received vaccines were also evaluated. The results showed that at 14 days after vaccination, none of the mouse groups that had received the vaccine generated IL-4 or IL-10. Furthermore, mice were given the pNZ8148-Usp45-omp25-L. lactis vaccine, and the IRBA vaccination showed significantly higher IL-4 and IL-10 titres at 28 days after immunization (p < 0.05) than were mice infected with control groups. In fact, pNZ8148-Usp45-omp25-L. lactis and IRBA vaccination groups' IL-4 and IL-10 titres were equivalent to and significantly higher than those in the PBS control group after 28 days (p < 0.05).
Additionally, a quantitative real-time PCR assay was used to deter- lactis and IRBA vaccination groups compared to other groups ( Figure 5).

Protective activity
Using virulent B. abortus strains to challenge the mice, the possible protective action brought on by various vaccination groups was assessed.
Three separate replicates of the experiment were run using the B. abortus strains. When compared to groups immunized with Ethe pNZ8148-Usp45-omp25-L. lactis and IRBA vaccines, the spleen weights of mice given various control vaccinations at 15 days after infection significantly decreased (Table 4). Mice were given the pNZ8148-Usp45-omp25-L. lactis and IRBA immunization groups showed no statistically significant changes (p > 0.05). The non-challenge groups, pNZ8148-Usp45-omp25-L. lactis and IRBA vaccination groups did not vary significantly (p > 0.05) from each other. Six mice exposed to B. abortus were randomly selected, and their mortality, weight changes and health status were monitored daily for 15 days. According to Table 4, all control group mice died 7 days following the test. After being challenged with a fatal dose of B. abortus isolates, the 15-day survival rates of mouse model vaccinated with the pNZ8148-Usp45-omp25-L. lactis were 87.5%, respectively. These numbers were noticeably higher than those of mice immunized with IRBA (50%). Each group's body mass and clinical symptom ratings dropped to their lowest levels 7 days after the challenge. The mice's body weight returned to normal 15 days after the experiment, and the symptoms disappeared.

Bacterial loads and pathological changes
Using a challenging experiment in BALB/c mice to gauge protection, we examined the effectiveness of the pNZ8148-Usp45-omp25-L. lactis immunization. All groups received three clinical B. abortus strain 2308 isolates. Six mice from each group were randomly selected to count the number of bacteria in the spleen tissue ( Figure 6A). Six mice from each group were taken 48 h after the challenge, serially diluted, and then plated on Brucella agar with 5% blood plates. The plates were next overnight incubated at 37 • C. The log10 CFU/mL value was calculated and compared following the CFU count. The spleen bacterial burdens were lower in the mice who got the pNZ8148-Usp45-omp25-L. lactis vaccine than they were in the animals in the other groups ( Figure 6B).
The challenge was followed by aseptic removal of the left spleen F I G U R E 5 (A) Cytokine levels were found in the small intestine of the control and immunized groups; (B) cytokine levels were found in the spleen of the control and immunized groups. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
tissue, preservation in a 4% formalin solution, haematoxylin-eosin staining and microscopic examination. Figure 7 displays that compared to other groups, the spleen portions from the pNZ8148-Usp45-omp25-L. lactis and IRIBA vac groups had less extensive spleen injuries, alveolar oedema, lymphocyte infiltration and morphological damage due to the inflammatory process. The pNZ8148-Usp45-omp25-L. lactis vaccine groups were more typical than the other groups, with cleaner alveoli. The amount of spleen failure in the pNZ8148-Usp45-omp25-L. lactis groups after mice were infected with the vaccine were considerably less severe than in the other groups, suggesting that the mice had less of an inflammatory response to spleen failure.

DISCUSSION
The high incidence of brucellosis as an economically significant human illness has prompted research into various tactics, such as creating subunit vaccines and using bacterial/viral vectors, to create more efficient vaccines (Dadar et al., 2021). To overcome the drawbacks of the currently utilized live vaccines, including attenuated pathogens, safer, more affordable and simpler to deliver vaccinations are still required.
E. coli is the most often utilized microbial host for the generation of protein. The majority of investigations utilized the E. coli BL21 host expression system to produce Brucella antigens such as omp10 and omp19, omp25, HSP and TF Proteins (Rezaei et al., 2020). However, an E. coli expression system does have certain limitations. In general, intracellular expression is one of the most widely utilized manufacturing techniques, although it is still expensive for industrial production. Pharmaceutical interests demand downstream purification procedures to remove endotoxin or harmful pyrogens from cell walls during manufacture (Singh et al., 2017). It has been demonstrated that L. lactis, a gram-positive lactic acid microorganism with a GRAS (Generally Regarded As Safe) status, has been utilized in food production for thousands of years (Díaz-Dinamarca et al., 2022). However, it is now used as a cell factory for producing heterologous proteins for therapeutic and commercial purposes and an intriguing alternative method for large-scale protein production (Jawan et al., 2021).
Many advantageous health qualities of L. lactis may be present.
According to Le Loir et al. (2005), this food-grade bacterium does not manufacture LPS, dose E. coli, form inclusion bodies, sporulate or move around much (Le Loir et al., 2005). It only has one membrane, which enables the investigation of the protein's function in entire cells. A disadvantage of using live LAB mucosal vaccines is the possibility of introducing genetically altered organisms with antibiotic resistance genes into the environment and host microflora. As a result, auxotrophic mutants and food-grade selection markers (such as bacteriocin resistance and generation), depending on the existence of these selectable markers on the plasmid of interest, could allay worries about the risk of unchecked release of nucleic acids into the environment by reducing the use of antibiotic resistance markers and harmful substances and potentially improve the applicability on an industrial level or in food products (He et al., 2012).
Additionally, L. lactis is a live delivery vehicle technology with several advantages over constitutive approaches, such as not being a commensal bacteria and not colonizing. Due to its immunomodulatory properties, ability to survive passage through the gastrointestinal tract, ease of uptake by M cells inducing potential immune reaction and single extracellular housekeeping protease, HrtA results in a very low rate of protein degradation, in vivo administration via oral administration and safer vaccination (Guillot et al., 2016). Only a few prior studies have examined the development and targeting of specific intracellular Brucella proteins in L. lactis, including L7/L12, GroEL heat-shock antigen and Cu, Zn superoxide dismutase (Sáez et al., 2012). Some brucellosis vaccines focus on discovering immunodominant proteins dependent on OMPs that can trigger a strong immune reaction. Due to its superior antigenicity, availability and surface-exposed loops predicted by thorough bioinformatics research to develop an effective vaccine in future research against brucellosis, the omp25 protein, one of the minor 25-kDa OMPs in Brucella spp., was chosen as an immunogenic candidate in the current study (Piri-Gharaghie, Beiranvand, et al., 2022;Piri-Gharaghie, Doosti, et al., 2022).
The omp25 protein we chose for our investigation stands out because, as a part of the exoproteome of the Brucella cell, it is surfaceexposed, accessible to monoclonal antibodies and may offer more significant benefits than other intracellular and periplasmic proteins that have been previously studied. Omp25's acceptable molecular weight (about 25 kDa) is also promising for excellent expression and an easy subsequent purification process. We hypothesized that the coadministration of immunomodulatory cytokines and antigens would boost the immunogenicity of the fusion protein (romp25-Usp45) and the efficacy of our constructed vaccine when Usp45 was expressed as an omp25 secretion factor of B. abortus in the future in vivo study on the topic of vaccination against brucellosis. This is the first research on the cloning, expression and purification of the romp25/Usp45 fusion protein. Prior research was conducted on the co-expression of secretion factor and antigen. Previous studies have shown that mice inoculated with bacterial species that simultaneously generated tetanus toxin fragment C (TTFC), IL-2 or IL-6 had more significant antibody titres for test TTFC (Gupta et al., 2020).
To test the notion, however, a different experiment that compares the immunity of omp16, whether fused or not, to IL-2 and performs in vivo study is needed. This study is the first to use a unique promoter to produce the recombinant omp25 polypeptide in L. lactis. Low pHinduced expression when cells were grown on glucose shift from the post-exponential to stationary phases (Zeng et al., 2022;Azadbakht et al., 2022). Without the use of costly or hazardous exogenous compounds and the ability to recover the protein quickly in subsequent steps, this induction is beneficial as an alternative and cost-effective tool for the production of heterologous proteins of therapeutic or technological interest. Some of the issues stem from synthesizing proteins using a pH-inducible promoter controlled by the extracellular level of ions like Cl or Zn (Peter et al., 2022). Consequently, expression optimization should be carried out to enable high-yield manufacturing.
It is essential to highlight that while though B. abortus recombinant food grad vaccines have highly positive outcomes in mouse models; the protective immunity shown in these models could not accurately represent the level of protection attained in wild hosts like cattle following immunization (Corner et al., 2010;Kargar et al., 2014). Before administering to cattle, more research evaluating protective effects in animal studies such as rats, guinea pigs and monkeys is thus urged. Recombinant vaccines are economically inappropriate for immunizing cattle because they require numerous booster injections, adjuvants and a mix of various antigens Dorneles, Teixeira-Carvalho, et al., 2015). Therefore, to make these vaccines inexpensive for widespread use, it is necessary to lower the cost of manufacturing, look for efficient and affordable adjuvants and reduce the cost of recombinant protein purification.

CONCLUSIONS
As a result, our study offers a novel method for using the food-grade, non-pathogenic and noncommercial bacterium L. lactis as a protein cell factory to produce the novel immunogenic fusion candidate romp25.
This method offers an appealing new approach to assessing the costeffective, safe, sustainable, simple pilot development of pharmaceutical products. It suggests a workable method for delivering additional heterologous protein antigens. We have already started doing additional studies in our labs, such as testing the immunogenicity and protective effectiveness of this recombinant L. lactis-romp25 strain as a potential oral vaccine candidate vs. virulent Brucella infection challenge in mice.

AUTHOR CONTRIBUTIONS
Somaye Tirbakhsh Gouran conducted research and drafted the manuscript; Abbas Doosti conceived and designed research and made manuscript revision; Mohammad Saeid Jami designed research and analysed data; Somaye Tirbakhsh Gouran provided funding support. All authors read and approved the final manuscript.